Mechanisms of immunopathology of COVID-19/ARDS, and strategies to mitigate detrimental inflammatory responses

National Institute Of Allergy And Infectious Diseases

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Abstract

The SARS-CoV-2 global pandemic and the possible risk of emerging human transmission of H5N1 influenza (bird flu) has emphasized the need for better understanding of the pathobiology of lethal viral pneumonias to enable the development of therapeutics to complement success in vaccination. To date, our attempts to develop and employ mouse models of SARS-Cov2 infection that cause death by pulmonary damage have not been successful, despite establishing 12 models of SARS-CoV-2 infection in 10 strains of mice. The 10 strains of mice used included the 8 founder strains of the Collaborative Cross (B6, A/J, 129SJ, NZO, NOD, PWK, CAST and WSB) with additional strains Balb/c and DSB. Together, these strains represent over 90% of the genetic diversity within Mus musculus and have enabled us to model distinct disease phenotypes including a) sensitive mice with high sustained virus replication in lung and CNS (B6,A/J), b) resistant mouse strains associated with lower peak virus titer and earlier control of replication in the lung with no or low dissemination to other organs (PWK, NZO), and c) sex bias where resistance is independent of virus titer in the lung suggesting a sex-differences in host response (CAST, NOD, WSB). The work was published (PMID: 37491352) and additional follow up data is being analyzed including RNAseq data from lungs and brain of all models, as well as additional spatial transcriptomics of lungs. Because of these findings, we have therefore focused our efforts on an updated model of lethal mouse influenza mediated by the PR8 H1N1 strain. During the past year we have completed studies involving (i) tests of interventions in the lethal influenza model that might have clinical utility and (ii) molecular, cell, and tissue level studies aimed at better understanding the underlying mechanism(s) of tissue damage and why interventions that constrain viral replication or innate immunity often fail after an early point in infection but well before death of the host. Using a severe influenza infection model that bypasses early nasopharyngeal replication and leads to rapid deep lung infection, we found that only very early treatment with the anti-viral oseltamivir phosphate (Tamiflu) could prevent death. Among 50 single or combined treatments covering many of the agents tested or used clinically for COVID-19 treatment (anti-IL-6, PANAM-G3, PMX205, inosine Pranobex, anti-PSGL1, ruxolitinib, inbrutinib, acalabrutinib, dypridamole, baricitinib, colchicine, silvelestat, AZD5059, anti-IL-6R, anti-CCL2, and Zileuton among others), none reduced weight loss or led to survival of more than a single infected animal, and several worsened disease. These findings argue that either (i) multiple damaging activities are involved and blunting only one is insufficient for a clinical effect, a possible but unlikely explanation as several of the tested drugs act broadly on innate immune responses and cytokine production/response. and/or (ii) that irreversible tissue damage occurs early and once this occurs, interfering independently with viral replication or host immunity does not play a major role in preventing eventual death. Imaging of whole lung lobes using our IBEX method for multiplex staining showed that in this influenza infection model, there was early infiltration by neutrophils, extensive spread of the virus, loss of pro-surfactant and associated type 2 pneumocytes, alveolar disruption, and myeloid cell bronchiolar plugging, followed by later arrival of T cells in concert with marked loss of viable lung tissue. While some treatments modified the balance and extent of cell infiltrates, none prevented the damage and parenchymal loss. From these data, we developed the hypothesis that the infected animals rapidly pass a tipping point with respect to residual functional pulmonary capacity and that after this point, interference with inflammatory processes alone is insufficient to rescue the animals. This led to a change in strategy based on combining arrest of further damage and promoting recovery of functional lung structures by enabling more effective repair of damaged pulmonary epithelium. Results from other laboratories indicated that type 1 interferons can inhibit pneumocyte proliferation, while other studies suggest that late arrival of cytotoxic T cells in the lung can cause loss of residual functional epithelial cells. We therefore combined low dose Tamiflu administered late in the course of infection in combination with one of two additional treatments that either promote pneumocyte replication (blocking of interferon signaling) or limit further immune destruction (depletion of CD8+ T cells) and found that each of these combination approaches can markedly reduce the frequency of death among infected animals. Imaging and RNA sequencing showed that the anti-IFNR treatment led to the anticipated improvement in lung repair with proliferation of epithelial cells associated with expression of genes in basal epithelial cells whose expression increases in the lung following injury, while keratin (KRT) 14+ p63+ KRT5+ cells were enhanced and acted as progenitors to damaged epithelial layers. We also observed increased pSTAT3 in AT2 cells and STAT3 signaling in these cells is known to promote alveolar epithelial regeneration via the brain-derived neurotrophic factor (BDNF) - tropomyosin-related kinase receptor B (TrkB) axis. The CD8 T cell depletion prevented further rapid impairment of lung function by destruction of viable but infected pneumocytes and maintenance of a higher frequency of surfactant positive cells. These findings provide a new perspective on why anti-inflammatory therapies have been largely unsuccessful in ameliorating late severe COVID-19 and suggest that late use of anti-virals typically recommend for use only in the very early phase of infection could, if combined with treatments that facilitate repair or prevent late adaptive immune damage, provide a new approach to treatment of patients hospitalized due to severe pulmonary viral pneumonia. These findings are reported in a paper in the journal Science.